Method for preparing malonate methylidene nanoparticles, nanoparticles optionally containing one or several biologically active molecules

ABSTRACT

The invention relates to a method for the preparation of nanoparticles formed from a random polymer of at least one compound of formula (I)                    
     in which 
     A represents a                    
      group or a                    
      group; 
     R 1  and R 2 , identical or different, represents a linear or branched C 1 -C 6  alkyl group; 
     n=1, 2, 3, 4 or 5, 
     characterised in that the monomer is dissolved beforehand in a water-miscible aprotic organic solvent forming, with the polymerisation medium, a non-solvent mixture of the polymer formed.

BACKGROUND OF THE INVENTION

The present invention relates to a novel method for the preparation ofnanoparticles formed from a polymerised methylidene malonate compound,said nanoparticles, optionally containing one or more biologicallyactive molecules, as well as to pharmaceutical compositions containingthem.

“Nanoparticles” is understood as meaning sub-micron particles having adiameter of less than about 500 nanometres. Nanoparticles formed byemulsion polymerisation of an alkyl cyanoacrylate are described in theEP 0 007 895 patent. The method used in the preparation of these alkylcyanoacrylate particles relies on the (anionic) polymerisation of themonomer which takes place spontaneously and in an aqueous medium. Thepreparation which follows the same principle (anionic emulsionpolymerisation) of nanoparticles constituted of a methylidene malonatepolymer is described notably in F. Lescure et al, Pharm. Res., 1994.11L1270-1276. These monomers, whose preparation is described in the EP 0283 364 patent, have a structure close to that of the cyanoacrylates butthe nitrile function of the latter is replaced with an ester or an esterester. Like the cyanoacrylates, they polymerise in the cold in anaqueous medium and can be biodegradable.

However, the methylidene malonate nanoparticles thus obtained possesscertain drawbacks.

In fact, the emulsion polymerisation of methylidene malonates in theform of nanoparticles leads, in aqueous phase and at slightly acid pH,to the formation of oligomers, mainly of the trimer or tetramer type,which are highly biodegradable.

These molecular species are partially hydrosoluble, such that thedispersion of these nanoparticles in an aqueous medium leads to theirsolubilisation and to the rapid loss of the particle structure (P.Breton et al., Eur. J. Pharm. Biopharm., 1996, 47, 95-103). When abiologically active molecule is associated with the methylidene malonatenanoparticles, is therefore possible for the molecule to be releasedvery rapidly after the administration, following the effect of dilutionin the circulatory current which brings about the rapid solubilisationof the oligomers which form the particle matrix, before eventuallyarriving at the site of action of the active principle.

Certain experiments have shown that the polymerisation at basic pHenabled the formation of polymers of higher molecular masses whilemaintaining the size of the nanoparticles. However, such syntheses arecharacterised by:

the impossibility of obtaining polymers of Mw<10000, and a fortioriMw<8000, constituting individualised nanoparticles without formingaggregates and without the significant presence of oligomeric species.

the impossibility of constituting polymers of Mw>20000 and a fortiori ofhigher Mw, at high pH (pH>7) without the inevitable formation ofaggregates which render the intravascular administration of thesepreparations impossible.

“Mw” is understood as meaning the mass average molecular mass (oraverage molecular mass) defined as: Mw=Σni. Mi²/Σni. Mi and Mp means themolecular mass of the quantitatively major species.

In the rest of the description, the molecular mass is expressed inpolystyrene equivalents (Ep).

This preparative method is therefore not suitable if it is desired toprepare methylidene malonate nanoparticles constituted of:

polymers of average molecular mass between about 5000 and 10000, notablyabout 8000,

polymers of average molecular mass greater than 20000, without formingaggregates.

SUMMARY OF THE INVENTION

The present invention therefore consists of the preparation ofmethylidene malonate nanoparticles having a diameter of less than 500nm, in particular 100 to 500 nm, formed from homogeneous molecularspecies of wide-ranging masses (Mw between about 2000 and 80000). Theprinciple consists in dissolving the monomer in a water-miscible aproticorganic phase but which, under the conditions of preparation of thenanoparticles, forms, with the aqueous polymerisation medium, anon-solvent mixture of the polymer formed.

“Aprotic organic phase” or “aprotic organic solvent”, is understood asmeaning an organic phase or a solvent without labile proton which iscapable of initiating an anion.

DETAILED DESCRIPTION OF THE INVENTION

The advantages of this preparative method according to the invention arenumerous:

it enables a more homogeneous dispersion of the monomer in thepolymerisation medium,

it makes use of non-chlorinated solvents which are easy to evaporatesince they are volatile,

it prevents the formation of polymer aggregates,

it gives rise to high polymerisation yields

it enables the constitution of polymers of homogeneous wide-rangingmolecular mass (Mw about 2000 to 100000, notably about 2000 to 80000) informing nanoparticles having a diameter of less than 500 nm.

Furthermore, the method enables the use of dispersing agents such asnon-ionic surfactants or colloid protecting polymers, which leads toparticles having flexible surface properties.

Finally, the molecular mass of the oligomers/polymers which form thenanoparticles according to the invention can be perfectly mastered byadjusting the following preparative conditions:

the monomer concentration in the organic phase,

the pH and the molarity of the polymerisation medium,

the nature and the concentration of the dispersing agent,

the volume ratio of the aqueous phase (polymerisation medium)/organicphase,

the mode of introduction of the organic mixture in the aqueous phase.

In a 1^(st) aspect therefore, the invention relates to a method for thepreparation of nanoparticles formed from a random polymer of at leastone compound of formula (I)

in which

A represents a

 group or a

 group;

R₁ and R₂, identical or different, represent a linear or branched C₁-C₆alkyl group;

n=1, 2, 3, 4 or 5;

characterised in that the monomer(s) is (are), before thepolymerisation. dissolved in a water-miscible aprotic organic solventforming, with the polymerisation medium, a non-solvent mixture of thepolymer formed.

In an advantageous aspect, the invention relates to a method for thepreparation of nanoparticles formed from a polymer of a compound offormula (I)

in which

A represents a

 group or a

 group;

R₁ and R₂, identical or different, represent a linear or branched C₁-C₆alkyl group; n=1, 2, 3, 4 or 5;

characterised in that before the polymerisation, the monomer isdissolved in a water-miscible aprotic organic solvent forming, with thepolymerisation medium, a non-solvent mixture of the polymer formed.

According to a particular aspect, the method according to the inventionenables the preparation of nanoparticles having a diameter of less than500 nm, preferably between 100 and 500 nm, and an average molecular mass(Mw) between about 1000 and 100000, notably between about 1000 and80000, in particular between about 2000 and 80000, preferably betweenabout 8000 and 80000.

In particular, the method according to the invention comprises the stepsconsisting in:

preparing a solution of at least one compound of formula (I) in awater-miscible aprotic organic solvent.

adding, with stirring, this organic phase to an aqueous polymerisationmedium at a pH between 4.5 and 10,

recovering the nanoparticles thus obtained after homogenisation of themixture and evaporating the organic solvent in vacuo.

The aqueous polymerisation medium can also be added to the organic phasewhich contains the monomer dissolved beforehand, and according toanother aspect, the method according to the invention comprises thesteps consisting in:

preparing a solution of at least one compound of formula (1) in awater-miscible aprotic organic solvent,

adding, with stirring, to this organic phase an aqueous polymerisationmedium at a pH between 4.5 and 10,

recovering the nanoparticles thus obtained after homogenisation of themixture and evaporating the organic solvent in vacuo.

As illustrated later on in the Examples, the pH of the polymerisationmedium is selected as a function of the molecular mass of the polymerthat is desired to prepare.

Advantageously, the mixture of the organic phase and the aqueous mediumis homogenised by continuous stirring for about 30 minutes and then,optionally, the preparation is completed by distilled water.

The polymer formed precipitates in the polymerisation medium and can berecovered by filtration for example. The nanoparticle suspension thusobtained can then be conditioned and lyophilised.

The aprotic organic solvent used for dispersing the monomer(s) must be asolvent of said monomer(s) which should also be miscible with water.This solvent is preferably selected from acetone, acetonitrile, dioxaneand tetrahydrofuran, acetone being particularly preferred.

Preferred aspects of the method are the following:

the concentration of monomer(s) of formula (I) in the organic solvent isof the order 30 mg/ml to 150 mg/ml;

the molarity of the polymerisation medium is of the order of {fraction(1/30)} M to

volume ratio of the aqueous phase to the organic phase is between 3/1and 20/1, preferably between 3/1 and 15/1.

Advantageously, the polymerisation medium contains one or moresurfactants or colloid protectors.

The surfactants can be ionic or non-ionic surfactants for example.Non-ionic surfactants will preferably be used which are selected fromcopolymers of polyoxyethylene and polyoxypropylene, poloxamers andpolysorbates. As colloid protector agents, polysaccharide derivativeswill preferably be used, such as dextrans, hydrosoluble cellulosederivatives: polyethylene glycols: poly(vinyl alcohol).

Preferably, the compound polymerised to form the nanoparticles accordingto the method of the invention is a compound of formula (1) in which: Arepresents a

group, n=1 and R₁=R₂=ethyl.

In another preferred aspect, the compound polymerised to form thenanoparticles according to the method of the invention is a compound offormula (I) in which: A represents a

group, and R₁=R₂=propyl.

Advantageously, a mixture of compounds of formula (I) in which A is a

group or a

group as defined above, can also be random polyrnerised.

In a 2^(nd) aspect, the invention relates to the nanoparticles formedfrom a random polymer of at least one methylidene malonate compound offormula (I), having a diameter of less than 500 nm, preferably between100 and 500 nm and an average molecular mass (Mw) between about 1000 and100000, notably between 1000 and 80000, in particular between about 2000and 80000, preferably between about 8000 and 80000, obtainable by thismethod.

In particular, said nanoparticles, obtainable by this method, are formedfrom a polymer of a compound of formula (I), have a diameter of lessthan 500 nm, preferably between 100 and 500 nm and an Mw between about1000 and 80000, in particular between about 2000 and 80000, preferablybetween about 8000 and 80000.

In a preferred aspect, the invention relates to nanoparticles formedfrom a random polymer of at least one compound of formula (1), having adiameter of less than 500 nm, preferably between 100 and 500 nm and anaverage molecular mass (Mw) between about 8000 and 100000, preferablybetween about 8000 and 80000.

In particular, the invention relates to nanoparticles formed from apolymer of a compound of formula (1), having a diameter of less than 500nm, preferably between 100 and 500 nm and an average molecular mass (Mw)between about 8000 and 80000.

Advantageously, said nanoparticles are formed from a compound of formula(I) in which A represents a

group, n=1 and R₁=R₂=ethyl.

In another preferred aspects said nanoparticles are formed from acompound of formula (I) in which A represents a

group and R₁=R₂=propyl.

Advantageously, said nanoparticles can be constituted of a randompolymer of a mixture of compounds of formula (I) in which A is a

group or a

group as defined above.

According to a further aspect of the invention, said nanoparticlescomprise, in their polymeric network, one or more biologically activemolecules such as mentioned above.

In fact, in an advantageous aspect of the method according to theinvention, the organic phase (when it is a biologically active moleculewhich is insoluble in water) or the polymerisation medium can containone or more biologically active molecules.

“Biologically active molecule” is understood as meaning, in anon-limiting way, any molecule or macromolecule which has a prophylacticor curative biological activity, in vitro or in vivo, notably ananti-infectious agent, in particular an antiseptic agent, an antibiotic,an antiviral, an antiparasitic or antimitotic agent, notably ananticancer agent.

Antibiotic or antiseptic agents which can be used can be, for example,rifampicin and colistin.

As antiviral agents, didanosin, ribavirin, zidovudin, acyclovir,ganciclovir, foscarnet, vidarabin and zalcitabin can be cited in anon-limiting way.

Cis-plastin, 5-fluorouracil or taxol can, for example, be used asanti-cancer agents. Another advantageous antitumor agent is creatinephosphate whose activity is described in the application EP 0 614 366.

The invention also relates to pharmaceutical compositions containingsaid nanoparticles which comprise one or more biologically activemolecules in association with a pharmaceutically acceptable vehicle.

The compositions according to the invention can be compositions whichcan be administered for example orally, sublingually, subcutaneously,intramuscularly, intravenously, transdermally, locally, rectally, viathe pulmonary route, or nasally.

The suitable forms of administration notably comprise oral forms, suchas tablets, gelatine capsules, powders, granules and oral solutions orsuspensions, sublingual and buccal administration forms, as well assubcutaneous, intramuscular, intravenous, intranasal or intraocular andrectal administration forms.

The invention is illustrated by the Examples below, in which thepreparation of the particles is carried out at ambient temperature(about 21° C.). The size, or diameter, of the nanoparticles was measuredwith a laser diffusion counter (Coulter Electronic Inc., USA). Themolecular mass of the polymers was determined by gel permeationchromatography.

EXAMPLE 1

500 mg of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonylethene(laboratoires UPSA/CARPIBEM, France), already desorbed of SO₂ for 3hours under 25 mbars, are dissolved in 5.55 ml acetone. This solution isthen mixed gradually and under magnetic stirring with 50 ml of anaqueous medium buffered at pH 8 (Na₂HPO₄/KH₂PO₄. {fraction (1/15)} M)and containing 500 mg of dextran 70 (FLUKA CHEMIE, Switzerland). Thealmost instant polymerisation produces a cloudiness of the mixture whichpossesses a Tyndall effect characteristic of colloidal solutions.Stirring is maintained for 30 minutes after the complete introduction ofthe organic phase. Next, 50 ml of distilled water containing 2.5 g ofglucose or trehalose (colloid protectors and cryoprotectors) are addedto the nanoparticle suspension and the mixture is submitted to anevaporation in vacuo so as to remove the acetone and to reduce thevolume of the aqueous suspension to 50 ml. After filtration on filterpaper (pore diameter 5 to 15 μm). the preparation is lyophilised. Asmeasured by laser diffusion, the particles contained in the filtratehave a diameter of 288 nm. The average molecular mass (Mw) of themethylidene malonate constituting the polymer matrix of the particles isevaluated to be 67000 by gel permeation chromatography.

EXAMPLE 2

pH Variation Study.

The experiment is carried out following the technique described inExample 1, but only varying the pH only of the phosphate buffer. Theresults are given in Table 1 below, in which Mp is the molecular mass ofthe principal species and Mw is the average molecular mass of thepolymer.

TABLE 1 pH of the polymerisation medium 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0size (nm) 280 344 424 423 361 382 313 288 standard deviation +/− nm 9 97 6 9 7 2 3 characteristics of the polymer (Ep) Mp 662 655 655 1970031500 36900 40300 59300 Mw 2080 4740 11140 17600 28900 39000 53200 67200

The results show that the average molecular mass of the polymers whichconstitute the nanoparticles increase regularly with the pH of thepolymerisation medium.

The gel permeation chromatographic profile of FIG. 1 represents thedistribution of the molecular mass of the polymer prepared at pH 5.5(concentration: 90 mg/ml). A broad peak is observed in 1 whichcorresponds to the species of high average molecular mass (Mw) and anarrow peak is observed in 2 which corresponds to the minor oligomers(major trimers and tetramers).

The dotted lines limit the analysable portion of the chromatogram. PeakF is that of toluene used as internal standard and the negative peakcorrespond to traces of water.

EXAMPLE 3

Study of the Variation of the Monomer Concentration.

The experiment is carried out following the technique described inExample 1. but by varying only the monomer concentration in acetone. Theresults are given in Table 2 below:

TABLE 2 monomer concentration in the organic phase (mg/ml) 30 60 90 size(nm) 213 239 288 standard deviation 2 4 3 +/−nm characteristics of thepolymer (Ep) Mp 31500 39600 59300 Mw 44700 63000 67200

The results show that the molecular mass of the principal species (Mp),as well as the average molecular mass (Mw) of the polymers whichconstitute the nanoparticles, increase regularly with the concentrationof the monomer in the organic phase.

EXAMPLE 4

The experiment is carried out according to Examples 1 to 3 but inreplacing dextran 70 colloid protector with a non-ionic surfactant,Pluronic F68 (BASF Corporation, USA).

The results are given in Table 3 below.

TABLE 3 pH of the polymerisation medium containing 0.5% Pluronic F 684.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Size (nm) 87 80 95 117 122 121 146 153standard deviation +/− nm 1 2 2 5 9 1 3 1 characteristics of the polymer(Ep) * Mp 656 13300 14800 25600 38600 43700 45300 77800 Mw 5520 974012300 23600 33000 51600 70900 88900 * concentration of the monomer inacetone = 90 mg/ml.

The results show, for the same conditions of pH:

an increase in the molecular mass of the principal species (Mp) and inthe average molecular mass (Mw) of the polymers constituting thenanoparticles in the presence of the surfactant with respect to thecolloid protector,

a decrease in the size of these same nanoparticles in the presence ofthe surfactant with respect to the colloid protector.

EXAMPLE 5

Study of the Molarity of the Polymerisation Medium

According to the method described in Example 1,500 mg of monomer aredissolved in 16.6 ml acetone are introduced into a phosphate buffer(Na₂HPO₄/KH₂PO₄) of increasing molarity, and further containing 0.5%Pluronic F68.

The results are given in Table 4 below:

TABLE 4 standard size of the deviation Mp Mw molarity nanoparticles (nm)(nm) (Ep) (Ep) 0.033M 127 2 15200 12500 0.066M 123 1 14600 12400 0.133M124 1 653 9790 0.267M 179 3 660 8690

The results show a decrease in the average molecular mass (Mw) of thepolymers which constitute the nanoparticles in proportion to an increasein the molarity of the medium.

EXAMPLE 6

Nanoparticles are prepared according to Examples 1 to 3 and are comparedto the nanoparticles prepared according to the method described byLescure et al., Pharm. Res. 1994. 11, 1270-1276. For this, 100 mg ofmonomer are introduced with stirring in 10 ml of a phosphate buffermedium (Na₂HPO₄/KH₂PO₄. {fraction (1/15)}M) of pH 5 to 8.

The results are given in Table 5 below in which the oligomers aredefined as any molecular species of molecular mass less than or equal to920.

TABLE 5 pH of the polymerisation medium containing 1% dextran 70 5.5 6.06.5 7.0 7.5 8.0 Method according size (nm) 260 296 337 335  271  322 toLescure et standard 5 5 10  4   6   6 al., 1994 deviation +/ − nm Mp 666660 675 685 15572 13000 Mw 1719 2421 5335 6041*  6759*  7594* %oligomers 43 53 27  38   18   13 yield of nano- 87 79.5 71.5   59.5   21  23 particles obtained % ± 5 Method according size (nm) 424 423 361 382 313  288 to the invention** standard 7 6 9  7   2   3 deviation +/ − nmMp 655 19695 31508 36290  40278 59300 Mw 11138 17569 28918 38997  5318167201 % oligomers 19 14 8  4   3   2 yield of nano- 82.5 73 84.5  91  85    86.5 particles obtained % ± 5 *presence of aggregates **monomerconcentration in acetone = 90 mg/ml

The results show that, for any experimental condition of identical pH:

the average molecular mass (Mw) of the polymers constituting thenanoparticles prepared according to Lescure et al. is less than that ofthe polymer obtained according to the method of the invention,

the contents of the oligomers (trimers-tetramers) constituting thepolymers are significantly less for the nanoparticles prepared accordingto the method of the invention;

the yields of polymerisation in the form of nanoparticles are higher forthe method of the invention compared to the method according to Lescureet al (the formation of aggregates results in low yields at basic pH forthe method according to Lescure et al.).

The gel permeation chromatography profile of FIG. 2 represents thedistributions of molecular mass of the polymers prepared at pH 7.5according to the method of the invention on the one hand (trace A), andaccording to the method of Lescure et al on the other (trace B). Apartfrom peak 3 corresponding to toluene, for peak A, a single peak 1 isobserved which corresponds to the principal species (Mp=40278) while fortrace B, the presence of a significant peak 2 is observed also whichcorresponds to the oligomers (trimers and tetramers).

EXAMPLE 7

50 ml of an aqueous medium buffered at pH 5; 6.5 or 8 (Na₂HPO₄/KH₂PO₄{fraction (1/15)}M) and containing 0.5% of Pluronic F68 (BASFCorporation, USA) are added gradually and with magnetic stirring to 5.55ml of a solution of 500 mg of1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxy-carbonylethene monomer(LABORATOIRES UPSA/CARPIBEM, France), already desorbed of SO₂ for 3hours under 25 mbars, in 5.55 ml of acetone. The stirring is maintainedfor 16 hours for the tests at pH 5 and 6.5 or for 30 minutes for thetest at pH 8 after the complete introduction of the organic phase. Next,50 ml of distilled water containing 2.5 g of glucose or trehalose(colloid protectors and cryoprotectors) are added to the nanoparticlesuspension and the mixture is submitted to evaporation in vacuo so as toremove the acetone and to reduce the volume of the aqueous suspension to50 ml. After filtration on filter paper (pore diameter 5 to 15 μm), thepreparation is lyophilised. The diameter of the particles contained inthe filtrate is measured by laser diffusion. The average molecular mass(Mw) of the methylidene malonate constituting the polymer matrix of theparticles is evaluated by gel permeation chromatography.

The results are given in Table 6 below, in which Mp is the molecularmass of the principal species and Mw is the average molecular mass ofthe polymer.

The yield is determined by the ratio of the amount of monomer introducedinto the reaction medium and the amount of polymer constituting thenanoparticles.

TABLE 6 pH of the polymerisation medium 5.0 6.5 8.0 size (nm) 848 394754 standard deviation 36 32 34 +/−nm characteristics of the polymer(Ep) Mp 312 24300 26500 Mw 6450 20100 20100 yield % 59 57 30 standarddeviation 5.1 4.6 4.2

EXAMPLE 8

Use of Different Solvents.

The experiment is carried out following the method of Example 1, butusing acetone, acetonitrile or tetrahydrofuran (THF) as solvent of themonomer.

The results are given in Table 7 below.

TABLE 7 Average particle Solvent size (nm) yield (%) Mw Acetone 253 7454 100 Acetonitrile 197 69 31 700 THF 191 70 30 300

EXAMPLE 9

Study of the Water/Solvent Volume Ratio

The experiment is carried out following the method of Example 1, butvarying the water/acetone volume ratio.

The results are given in Table 8 below:

TABLE 8 Water/solvent volume ratio 4.5/1 9/1 18/1 size (nm) 241 288 334yield (%) 74 74 85 characteristics of the polymer Mp 62100 59300 33100Mw 42000 67200 24600

EXAMPLE 10

Implementation of the method at pH 10.

The tests were carried out in an aqueous medium at pH=10 in the presenceeither of a surfactant or a colloid protector and this, either followingthe method of Example 1 or following the method of Example 7.

1) test 1

100 mg of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonylethenemonomer are dissolved in 1 ml of acetone.

This solution is then added gradually and with magnetic stirring into 10ml of an aqueous medium at pH=10 and containing 100 mg of Dextran 70.

The polymerisation is instantaneous. The stirring is maintained for 30minutes after the introduction of the whole of the organic phase. Next,10 ml of distilled water are added to the nanoparticle suspension, andthe mixture is submitted to an evaporation in vacuo so as to remove theacetone. The medium is then centrifuged (v=10000 rpm, 10 min at 4° C.).

2) test 2

The experimental protocol is identical to that of test 1 but byreplacing Dextran 70 with Pluronic F68.

3) test 3

10 ml of an aqueous medium at pH=10 containing 100 mg of Dextran 70 areadded gradually with magnetic stirring into arr organic phaseconstituted of 100 mg of monomer and 1 ml of acetone. The polymerisationis instantaneous. The stirring is maintained for 30 minutes after theintroduction of the whole of the aqueous phase. Next, 10 ml of distilledwater are added to the nanoparticle suspension and the mixture issubmitted to an evaporation in vacuo so as to remove the acetone. Themedium is then centrifuged (v=10000 rpm, 10 min at 4° C.).

4) test 4

The experimental protocol is identical to that of test 3 but the Dextran70 is replaced with Pluronic F68. After centrifugation, thenanoparticles contained in the plug are analysed by steric exclusionchromatography to determine their weight average molecular mass (Mw).

The results are given in the Table 9 below.

TABLE 9 Mw Particle size (nm) Test 1 8 800 240 Test 2 6 900 245 Test 3 1400 316 Test 4 1 850 333

EXAMPLE 11

The experiment is carried out following the polymerisation techniquedescribed in Example 1, but using 1,1-propoxycarbonylethene(Laboratoires UPSA/CARPIBEM, France) hereinafter referred to as MM 3.3,alone or in a mixture with the1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxy-carbonylethene monomer(Laboratoires UPSA/CARPIBEM. France), hereinafter referred to as MM2.1.2. The results are given in Table 10 below, in which Mp is themolecular mass of the principal species and Mw is the average molecularmass of the polymer.

TABLE 10 Ratio MM 3.3/MM 2.1.2 100/0 75/25 50/50 25/75 Size 123 223 298155 Yield (%) 77 73 80 78 Characteristics of the polymer Mp 44764 9209037467 21727 Mw 44122 89793 37467 21727

EXAMPLE 12

Preparation of Nanoparticles Containing Rifampicin

5 mg of rifampicin base (Sigma) are dissolved in 1 ml of acetone towhich 90 mg of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxy-carbonylethene monomer (LABORATOIRES UPSA/CARPIBEM. France)are added, beforehand desorbed of SO₂ for 3 hours under 25 mbars. Withthe aid of a glass pipette, this solution is then added gradually andwith constant stirring (750 rpm) to 9 ml of aqueous medium buffered atpH 6.0 with the aid of a phosphate buffer (Na₂HPO₄/KH₂PO₄ 0.066M) andcontaining 90 mg of dextran 70 (1% w/v). After 18 hours ofpolymerisation at 20° C., 9 ml of distilled water containing 5% ofD-glucose are added with stirring to the nanoparticle suspension, themixture is then submitted to an evaporation in vacuo with the aid of aRotavapor (20° C., 25 mbars) so as to remove the acetone and to reducethe volume of the aqueous suspension to 9 ml. The preparation is thenlyophilised: freezing takes place at −30° C. and sublimation at +20° C.for 36 hours at a pressure of 0.05 mbar.

The size of the nanoparticles and the rifampicin concentration aremeasured before and after lyophilisation. The size is measured by laserdiffusion. The determination of the rifampicin is carried out by highperformance liquid chromatography coupled to a spectrophotometer. Themobile phase is composed of a mixture of methanol/0.05M ammonium acetate(65:35), the pH is adjusted to 7.3. the flow rate is fixed at 1 m/1 minand the absorption is read at 254 nm. The content of rifampicin which isnot bound to the nanoparticles is measured in the supernatant obtainedafter ultracentrifugation of the nanoparticle suspension (80000 g, 1 hat 4° C.). The amount of rifampicin bound to the nanoparticlescorresponds to the fraction present in the plug, which is dissolved inTHF before proceeding with the direct rifampicin determination.

The following results are obtained:

size of the nanoparticles containing rifampicin: 266±63 nm beforelyophilisation and 282±54 nm after lyophilisation;

percentage binding of rifampicin: 8.5±0.5% before and afterlyophilisation.

EXAMPLE 13

Preparation of nanoparticles containing colistin

The experiment is carried out in the same way as in Example 12, but theactive principle being hydrosoluble, it is incorporated in thepolymerisation medium at a concentration of 0.5 mg/ml before addition ofthe organic phase.

The size of the nanoparticles containing colistin measured by laserdiffusion is 282±65 nm after evaporation and 283±26 nm afterconservation at +4° C. for +4 days. Determined according to the gelosediffusion technique (S. P. Gotoff et al., Antimicrob. Agents Chemother,1962, 107-113), colistin is found at the concentration of 15 μg/ml inthe supernatant obtained after ultracentrifugation of the nanoparticlesuspension (80000 g, 1 hour at 4° C.): the fraction which is not boundto the nanoparticles is then evaluated at 3% of the total amount ofcolistin added.

EXAMPLE 14

Preparation of nanoparticles containing azidothymidine (AZT) (SigmaAldrich Chimie, France).

240 mg of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonyl-ethenemonomer (Laboratoires UPSA/CARPIBEM, France), already desorbed of SO₂for 3 hours under 25 mbars, are dissolved in 2.5 ml acetone. With theaid of a propipette, this solution is then gradually added and withconstant stirring to 22.5 ml of aqueous medium buffered at pH 8.0 withthe aid of a phosphate buffer (Na₂HPO₄/KH₂PO₄ 0.066M) and containing 225mg of dextran 70 (1% w/v), as well as the hydrosoluble active principleat a concentration of 0.53 mg/ml. After 18 hours' polymerisation at 20°C., 22.5 ml of demineralised water containing 5% of D-glucose are addedwith stirring to the nanoparticle suspension, the mixture is thensubmitted to an evaporation in vacuo with the aid of a Rotavapor (20°C., 25 mbars) so as to remove the acetone and to reduce the volume ofthe aqueous suspension to 39.0 ml. The preparation is then lyophilised;freezing takes place at −30° C. and sublimation at +20° C. for 36 hoursat a pressure of 0.05 mbar.

The size of the nanoparticles containing AZT measured by laser diffusionis 255±63 nm before lyophilisation. The content of AZT in thesupernatant after centrifugation of the nanoparticle suspension (12000rpm, 1 hour at 4° C.) is determined by UV spectrophotometry at 266 nm. Aconcentration of 98 μg/ml is obtained: the fraction which is not boundto the nanoparticles is therefore evaluated to be 31.9% of the totalamount of AZT added. The fraction of AZT bound to the nanoparticles istherefore 68.1%.

EXAMPLE 15

Preparation of Nanoparticles Containing Creatine Phosphate (BoehringerMannheim).

The encapsulation of creatine phosphate is carried out according to thetechnique of Example 14. The size of the nanoparticles containingcreatine phosphate measured by laser diffusion is 275±260 nm beforelyophilisation. The determination of the creatine phosphate is carriedout by high performance liquid chromatography coupled to aspectrophotometer. The mobile phase is composed of a phosphate buffer(KH₂PO₄. 0.05M) adjusted to pH 3.3. The flow rate is fixed at 2 ml/minand the absorption is read at 200 nm.

The content of creatine phosphate which is not bound to thenanoparticles is measured in the supernatant obtained aftercentrifugation of the nanoparticle suspension (12000 rpm, 1 hour at 4°C.). The creatine phosphate is found at a concentration of 463 μg/ml inthe supernatant: the fraction which is not bound to the nanoparticles istherefore evaluated at 81% of the total amount of creatine phosphateadded. The fraction of creatine phosphate bound to the nanoparticles istherefore 19%.

EXAMPLE 16

Preparation of Nanoparticles Containing 5-fluorouracile (5-FU)

The encapsulation of 5-FU (Sigma Aldrich Chimie, France) is carried outaccording to the technique of Example 14. The size of the nanoparticlescontaining the 5-FU measured by laser diffusion is 516±88 nm beforelyophilisation. Determined by UV spectrophotometry at 266 nm, the 5-FUis found at a concentration of 70 μg/ml in the supernatant obtainedafter centrifugation of the nanoparticle suspension (12 000 rpm, 1 hourat 4° C.): the fraction which is not bound to the nanoparticles istherefore evaluated at 23.3% of the total amount of 5-FU added. Thefraction of 5-FU bound to the nanoparticles is therefore 76.7%.

What is claimed is:
 1. A method for the preparation of nanoparticlesformed from a random polymer of at least one compound of formula (I)

in which A represents a

 group or a

 group; R₁ and R₂, identical or different, represent a linear orbranched C₁-C₆ alkyl group; n=1, 2, 3, 4 or 5, comprising the steps ofdissolving said at least one compound of formula (I) in a water-miscibleaprotic organic solvent, and mixing the dissolved at least one compoundof formula (I) with an aqueous polymerization medium, resulting in anon-solvent mixture of the polymer formed.
 2. The method according toclaim 1 for the preparation of nanoparticles formed from a polymer of acompound of formula (I)

in which: A represents a

 group or a

 group; R₁ and R₂, identical or different, represent a linear orbranched Cl-C₆ alkyl group; n=1, 2, 3, 4 or 5, comprising the steps ofdissolving said compound of formula (I) in a water-miscible aproticorganic solvent, and mixing the dissolved compound of formula (I) withan aqueous polymerization medium, resulting in a non-solvent mixture ofthe polymer formed.
 3. The method according to claim 1, for thepreparation of nanoparticles having a diameter of less than 500 nm, andan average molecular mass (Mw) between about 1000 and
 100000. 4. Themethod according to claim 1, wherein the steps comprise: preparing asolution of at least one compound of formula (I) in a water-miscibleaprotic organic solvent to form an organic phase, adding, with stirring,said organic phase to an aqueous polymerization medium at a pH between4.5 and 10 to form a mixture, and recovering the nanoparticles obtainedthereby after homogenizing the mixture and evaporating the organicsolvent in vacuo.
 5. The method according to claim 1, wherein the stepscomprise: preparing a solution of at least one compound of formula (I)in a water-miscible aprotic organic solvent to form an organic phase,adding, with stirring, to said organic phase, an aqueous polymerizationmedium at a pH between 4.5 and 10 to form a mixture, and recovering thenanoparticles obtained thereby after homogenizing the mixture andevaporating the organic solvent in vacuo.
 6. The method according toclaim 1, wherein the aprotic organic solvent is selected the groupconsisting of acetone, acetonitrile, dioxane and tetrahydrofuran.
 7. Themethod according to claim 1, wherein said at least one compound offormula (I) is present in the organic solvent in a concentration of theorder of 30 mg/ml to 150 mg/ml.
 8. The method according to claim 1,wherein the polymerization medium has a molarity of the order of{fraction (1/30)} M to ⅓ M.
 9. The method according to claim 1, whereinthe polymerization medium contains one or more surfactants or colloidprotectors.
 10. The method according to claim 9, wherein the surfactantsare non-ionic surfactants selected from the group consisting ofcopolymers of polyoxyethylene and polyoxypropylene, poloxamers andpolysorbates.
 11. The method according to claim 9, wherein the colloidprotectors are selected from the group consisting of dextrans,hydrosoluble cellulose derivatives, polyethylene glycols and poly(vinylalcohol).
 12. The method according to claim 1, wherein the organic phaseor the polymerization medium contains one or more biologically activemolecules.
 13. The method according to claim 1, wherein the polymerizedcompound is a compound of formula (I) in which A represents a

group, R₁=R₂=ethyl and n=1.
 14. The method according to claim 1, whereinthe polymerized compound is a compound of formula (I) in which Arepresents a

group and R₁=R₂=propyl.
 15. The method according to claim 1, wherein amixture of compounds of formula (I) in which A is a

group or a

group, is randomly polymerized.
 16. The method of claim 3, wherein thenanoparticles have a diameter between 100 and 500 nm.
 17. The method ofclaim 3, wherein the nanoparticles have an average molecular mass(M_(w)) between about 1000 and
 80000. 18. The method of claim 17,wherein the nanoparticles have an average molecular mass (M_(w)) betweenabout 2000 and
 80000. 19. The method of claim 18, wherein thenanoparticles have an average molecular mass (M_(w)) between about 8000and
 80000. 20. Nanoparticles formed from a random polymer of at leastone compound of formula (I)

in which A represents a

 group; R₁=R₂=propyl; and n=1, 2, 3, 4 or 5, having a diameter of lessthan 500 nm, and an average molecular mass (M_(w)) between about 8000and 100000, said nanoparticles being formed from homogenous molecularspecies.
 21. Nanoparticles according to claim 20, having a diameterbetween about 100 and 500 nm.
 22. Nanoparticles according to claim 20,having an average molecular mass (M_(w)) between about 8000 and 80000.